During the 5-year ELOXY project, a large effort was made to improve experimental procedures for oxygen-free cultivation of yeasts in laboratory systems. Using the resulting optimized systems, we established that, in contrast to a long-standing 'dogma' in yeast research, S. cerevisiae (bakers' yeast) is able to grow in the absence of oxygen without supplementation of unsaturated fatty acids. An absolute oxygen requirement of S. cerevisiae for synthesis of sterols and for the cofactors NAD and coenzyme A was confirmed and we discovered that synthesis of biotin by yeasts also requires oxygen.
Novel strategies to eliminate oxygen requirements in bakers' yeast were inspired by a fundamental research line on Neocallimastigomycetes ('Neo's'), a group of fungi that can grow in the complete absence of oxygen. During their evolution, Neo's acquired the ability to 'bypass' reactions that in other fungi and yeasts require oxygen. ELOXY focused on the mechanisms by which Neo's are able to synthesize the cofactors NAD and CoA in the absence of oxygen. Studying these processes by expressing candidate genes from these fungi in bakers' yeast was shown to be a powerful approach to understand evolutionary adaptations of Neo's, which are not yet accessible to genetic modification, to growth in oxygen-free environments.
Based on analysis of the genomes of Neo's, we were able to eliminate oxygen requirements of bakers' yeast for synthesis of the cofactors NAD and CoA by genetic modification. Evolutionary adaptations of Neo's to oxygen-free environments also provided the inspiration for a successful metabolic engineering strategy to bypass the oxygen requirements for sterol synthesis in baker's yeast by introducing a gene that enables production of tetrahymanol, a 'sterol surrogate' whose synthesis does not require oxygen. Introduction of a set of bacterial genes eliminated oxygen requirements for biotin synthesis. While these results provide clear proofs of principle for elimination of the main oxygen requirements of bakers' yeast under laboratory conditions, further research is required to translate them to robust industrial strains.
Research on sterol surrogates led to an unexpected discovery. The yeast Schizosaccharomyces japonicus was found to grow very fast in the absence of oxygen, sterols and unsaturated fatty acids. By a combination of genome analysis, chemical analysis of lipids in cells of this yeast and by expression of a single gene in bakers' yeast, it was shown that, during its evolution, Sch. japonicus acquired bacterial gene that enables it to make hopenes as sterol surrogates. The vigorous growth of Sch. japonicus in oxygen- and sterol-free media makes it a highly interesting model for further research on how membrane composition affects growth and robustness. Another unexpected finding concerned the synthesis of pyrimidines. In most yeasts and fungi, synthesis of these building blocks for DNA and RNA requires oxygen and only bakers' yeast and closely related yeasts were known to bypass this requirement. In the ELOXY project we showed that, during their evolution, Neo's, Schizosaccharomyces japonicus and the yeast Dekkera bruxellensis have, independently acquired another way to anaerobically synthesize pyrimidines. These results illustrate the flexibility of fungal evolution in natural environments.
A major effort was invested in analysing the oxygen requirements of two yeast species, Ogataea parapolymorpha and Kluyveromyces marxianus that, in contrast to bakers' yeast, can grow at temperatures of up to 50 degrees C, but cannot grow without oxygen. Early in the project, protocols were developed for CRISPR-Cas9-mediated genetic modification of these two yeasts. From a detailed quantitative analysis in bioreactor cultures, we found that O. parapolymorpha requires much more oxygen than K. marxianus. Analysis of product formation, combined with analysis of gene expression, revealed that the large oxygen requirements of O. parapolymorpha were related to multiple cellular processes, with regeneration of NAD as a major contributor. While genetic modification led to a reduction of the oxygen requirements, we were not yet able to completely eliminate them. Intensive studies on K. marxianus, encompassing bioreactor cultivation, genome and gene expression analysis and sterol-uptake studies strongly suggested that absence of a functional sterol uptake system was a key contribution to its inability to grow anaerobically. Inspired by our research on S. cerevisiae, we engineered K. marxianus for synthesis of the sterol surrogate tetrahymanol. This strategy, combined with laboratory evolution, yielded K. marxianus strains that grew in the absence of oxygen at a temperature of 45 degrees C. We hope to build on this breakthrough result to eventually enable the construction of robust K. marxianus strains that can be applied in high-temperature, cost-effective fermentation processes.
